Ovo vaccination of Campylobacter in avian species

ABSTRACT

The present invention provides a method of inducing an immune response against  Campylobacter  in an avian species, especially a domesticated avian species such as chicken, turkey, duck, goose and quail, by administering, in ovo, live cells of  Campylobacter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is filed under 35 USC 371 as a US National Stage PatentApplication of PCT/IB2004/003806, filed Nov. 22, 2004 under 35 USC 363,which claims the benefit under 35 USC 119(e)(1) of U.S. ProvisionalPatent Application No. 60/526,681, filed Dec. 3, 2003 under 35 USC111(b), the entire disclosures of which are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates to methods of inducing an immune response in anavian species against Campylobacter by administering, in ovo, liveCampylobacter cells.

BACKGROUND OF THE INVENTION

Consumption of poultry contaminated with Campylobacters has beenimplicated as a major source of human infection. Therefore, removal ofthese organisms from the poultry food chain has been a significantobjective of Campylobacter research.

Campylobacters typically grow and colonize in the avian gut environment.Colonization has been reported in chickens, ducks, pigeons, quail,ostriches and turkeys. Colonization of poultry does not result indisease but is commensal in nature.

The use of a competitive exclusion culture to exclude Salmonella orCampylobacter from the digestive tract of a bird has been described inU.S. Pat. No. 6,491,910. The efficacy of this method againstCampylobacter appears variable. In ovo vaccination with heat killedcells of Campylobacter jejuni has been described by Noor et al. (BritishPoultry Science, 1995. 36(4): 563-73) and by S. Noor (Jurnal IImu TernakDan Veteriner, 1998. 3(4): 264-269). In ovo immunization of chickenswith flagellin and whole cell protein antigens of Campylobacter jejunihas also been reported by S. Noor et al. (Jurnal IImu Ternak DanVeteriner, 2000. 5(2): 119-124). Efforts have also been made in order toidentify the genes involved in colonization (Ziprin et al., Abstracts,Poultry Science Association meeting, Aug. 8-11, 1999, Springdale, Ark.).See also, Rice, 1997, “Campylobacter jejuni in broiler chickens:colonization and humoral immunity following oral vaccination andexperimental infection”, Vaccine 15 (17-18): 1922-1932, wherein killedCampylobacter cells are administered; and Ziprin et al, 2002, CurrentMicrobiology 44(3): 221-223, wherein chicks were vaccinated post-hatchwith viable but non-colonizing strains.

Prior to the present invention, there has been no effective immunizationstrategy that employs in ovo administration with live Campylobactercells.

SUMMARY OF THE INVENTION

The present invention provides a method of inducing an immune responsein an avian species against Campylobacter by administering, in ovo, livecells of Campylobacter.

According to the present invention, live cells of Campylobacter can besafely administered to any avian species for the purpose of inducing animmune response against Campylobacter, especially a domesticated avianspecies such as chicken, turkey, duck, goose and quail.

Campylobacter species suitable for use in the present method include,but are not limited to, Campylobacter jejuni, Campylobacter coli,Campylobacter lari, or a combination thereof.

The cells of Campylobacter can be wild type cells or cells ofCampylobacter that have been genetically modified to contain one or moremutations in the genome, or to contain a desirable heterologoussequence.

Preferably, the cells are combined with a veterinary-acceptable carrierprior to administration and are administered in an amount that iseffective to induce an immune response in the avian species developedfrom the treated egg, preferably in an amount of at least about 5×10⁵,and more preferably, of at least about 1×10⁶ live cells.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly found in accordance with the present inventionthat live cells of Campylobacter can be safely administered in ovo, toeggs of an avian species, which results in effective colonization andinduces an immune response in the avian species against Campylobacter.

Accordingly, the present invention provides a method of inducing animmune response in an avian species against Campylobacter by in ovoadministration of live cells of Campylobacter.

As used herein, the term “avian species” or “bird” is meant to includeany avian species, including a domestic or a game bird, e.g., chicken,turkey, duck, goose, or quail. Preferably, live cells of Campylobacterare administered to eggs of a domesticated bird raised for commercialproduction of eggs or meat, such as a domesticated chicken, turkey,duck, goose and quail.

The term “Campylobacter” refers to any Campylobacter species or anystrain of a Campylobacter species, including Campylobacter jejuni,Campylobacter coli, Campylobacter lari. Campylobacter strains suitablefor use in the present method include C. jejuni UA535 and C. jejuni81-176, as well as the following mutant strains: C. jejuni CsrA (mutatedwithin gene Cj1103, a csrA homolog), C. jejuni HspR (Cj1230), C. jejuniHtrA (Cj1228c), C. jejuni Dps (Cj1534c), C. jejuni flbA (Cj0822c), C.jejuni Pnp (Cj1253), and C. jejuni SurE (Cj0293).

Both wild type and mutant strains of a Campylobacter species can be usedin the method of the present invention, including strains that have beengenetically engineered and contain one or more mutations in the genome.The capacity of a mutant Campylobacter strain in colonization andinduction of an immune response in an avian species can be determinedfollowing the techniques described in the examples hereinbelow ortechniques known to those skilled in the art.

In a preferred embodiment, the Campylobacter strain employed in themethod of the present invention has been genetically engineered tocontain a heterologous polynucleotide sequence. The heterologoussequence can be transferred into the Campylobacter strain via a plasmid,phage or cosmid vector by various means such as conjugation,electroporation, or transformation. Other methods such as transductionare also suitable, wherein the recombinant DNA in the form of atransducing phage or cosmid vector is packaged within a phage. Once therecombinant polynucleotide is in the carrier Campylobacter strain, itmay continue to exist as a separate autonomous replicon or it may insertinto the Campylobacter chromosome and be reproduced along with thechromosome during cell division.

According to the present invention, the heterologous polynucleotidesequence can encode an antigen from an organism, such as a virus,bacterium or parasite, that causes disease in bird or that causesfood-borne illness in humans upon consumption of a bird contaminatedwith the organism. Administration of live cells of geneticallyengineered Campylobacter containing such heterologous sequence caninduce an immune response in the avian species against bothCampylobacter and the pathogenic organism. Examples of such pathogenicorganisms include Salmonella, Escherichia coli, Eimeria, Clostridium,infectious bursal disease virus.

The heterologous polynucleotide sequence can also encode a proteinessential in colonization of domesticated birds by Campylobacter.Proteins known to be essential in colonization of Campylobacter include,e.g., the gene products of dnaJ and cadF.

Additionally, the heterologous polynucleotide sequence can encode aprotein or peptide that stimulates the immune system of bird. Examplesof proteins or peptides that stimulate the immune system of birdsinclude, e.g., cholera toxin or E. coli heat labile toxin.

Moreover, the heterologous polynucleotide sequence can itself enhancethe growth or feed efficiency of a domesticated bird, or can encode aprotein or peptide that enhances the growth or feed efficiency of adomesticated bird. Examples of such molecules or proteins includeepidermal growth factor, insulin-like growth factor, interleukins, andantimicrobial peptides.

According to the present invention, a Campylobacter species is culturedby standard methods known to those skilled in the art and live cells ofsuch Campylobacter are collected for in ovo administration to eggs of anavian species. Live cells of more than one Campylobacter species can becombined for in ovo administration.

In addition to Campylobacter cells, a veterinary-acceptable carrier canalso administered. Preferably, Campylobacter cells and aveterinary-acceptable carrier are both administered in ovo, eithertogether or separately. Alternatively, a veterinary-acceptable carriercan be administered to the bird any time post-hatch in feed or water, orby aerosol spray. The live Campylobacter cells administered, whetherwith or without a veterinary-acceptable carrier, are free ofneutralizing factors, such as neutralizing antibodies or fragments ofantibodies, as described in U.S. Pat. No. 6,440,408.

The term “a veterinary-acceptable carrier” includes solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antifungal agents, isotonic agents, adsorption delaying agents, and thelike. Diluents can include water, saline, dextrose, ethanol, glycerol,and the like. Isotonic agents can include sodium chloride, dextrose,mannitol, sorbitol, and lactose, among others. Stabilizers includealbumin, among others.

Adjuvants suitable for use in the present method include but are notlimited to: mineral gels, e.g., aluminum hydroxide; surface activesubstances such as lysolecithin; glycosides, e.g., saponin derivativessuch as Quil A or GPI-0100 (U.S. Pat. No. 5,977,081); cationicsurfactants such as DDA, pluronic polyols; polyanions; non-ionic blockpolymers, e.g., Pluronic F-127 (B.A.S.F., USA); peptides; mineral oils,e.g. Montanide ISA-50 (Seppic, Paris, France), carbopol, Amphigen(Hydronics, Omaha, Nebr. USA), Alhydrogel (Superfos Biosector,Frederikssund, Denmark) oil emulsions, e.g. an emulsion of mineral oilsuch as BayolF/Arlacel A and water, or an emulsion of vegetable oil,water and an emulsifier such as lecithin; alum, cholesterol, rmLT,cytokines and combinations thereof. The immunogenic component may alsobe incorporated into liposomes, or conjugated to polysaccharides and/orother polymers for use in a vaccine formulation. Additional substancesthat can be included in a product for use in the present methodsinclude, but are not limited to one or more preservatives such asdisodium or tetrasodium salt of ethylenediaminetetracetic acid (EDTA),merthiolate, and the like. Immunostimulants which enhance the immunesystem's response to antigens may also be included in a product.Examples of suitable immunostimulants include cytokines such as IL-12 orIL-2, or stimulatory molecules such as muramyl dipeptide,aminoquinolones, lipopolysaccharide, and the like. The adjuvant can becombined with live cells of Campylobacter prior to in ovoadministration, or can be administered independently any time afterhatching by way of feed, water or aerosol spray, provided to the bird.

The live cells are administered at a dose effective to induce an immuneresponse against Campylobacter in the avian species developed from thetreated egg. The amount of live cells of Campylobacter that is effectiveto induce an immune response, or “the immunizing effective amount”, mayvary, depending on the particular species or strains of Campylobacterused in the administration and the species of bird being immunized.Generally speaking, at least about 5×10⁵ live cells should beadministered to be effective in inducing an immune response. Morepreferably, at least about 1×10⁶ live cells of Campylobacter areadministered.

By “inducing an immune response” is meant that the live cells ofCampylobacter administered to an egg induce an immune response in thebird developed from the egg, which in turn provides some degree ofprotection to the bird against colonization of a Campylobacter species,which can be the same as, or different from, the Campylobacter speciesused in the in ovo administration.

An immune response induced by in ovo administration of live cells ofCampylobacter can be a cellular immune response mediated primarily bycytotoxic T-cells, or a humoral immune response mediated primarily byhelper T-cells, which in turn activates B-cells leading to antibodyproduction, or the combination of a cellular immune response and ahumoral immune response.

According to the present invention, one or more additional immunogenscan be included in the administration in ovo. Such immunogens includeantigens derived from viruses, e.g. avian infectious bronchitis virus,avian infectious bursal disease virus, avian encephalomyelitis virus,egg drop syndrome virus, influenza virus, reovirus, adenovirus,hydropericardium syndrome virus, among others; antigens derived frombacteria, e.g., Haemophilus paragallinarum, Salmonella typhimurium, S.enteritidis, S. pullori, S. gallinarum, S. choleraesuis, E. coli,Clostridium spp., Mycoplasma spp., enterococcus, among others; andantigens derived from protozoan, e.g. Eimeria tenella, E. maxima, E.acervulina, E. brunetti, E. necatrix, among others.

By “in ovo administration” is meant administration to eggs of an avianspecies, preferably eggs in the fourth quarter of incubation. That is,for chicken eggs, the administration is conducted preferably on aboutthe fifteenth to nineteenth day of incubation, and more preferably onabout the eighteenth day of incubation. For turkey eggs, theadministration is conducted preferably on about the twenty-first totwenty-sixth day of incubation, and more preferably on about thetwenty-fifth day of incubation.

The administration can be conducted by any method which results in theintroduction of live cells of Campylobacter into an egg through theshell. A preferred method of administration is by injection. Theinjection can be made at any site of an egg, so long as the injectiondoes not damage the tissues or organs of the embryo, or theextraembryonic membranes surrounding the embryo. The injection can beachieved by using any one of the well-known egg injection devices, suchas a conventional hypodermic syringe fitted with a needle of about 18 to22 gauge, or a high speed automated egg injection system as described inU.S. Pat. Nos. 4,681,063, 4,040,388, 4,469,047, and 4,593,646.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1

Broiler chicken eggs were injected in ovo on day 18 of incubation with10⁶ colony forming units (CFU) of Campylobacter jejuni UA535. This is awild type, unmodified strain of C. jejuni, originally isolated from ahuman clinical case of campylobacteriosis. A second group of eggsremained uninoculated until the day of hatch, at which time the chicksin this group were inoculated per os with 10⁶ CFU C. jejuni UA535. Athatch, the number of live chicks and unhatched eggs in each treatmentwere recorded and all chicks were placed on litter with 3 replicate pensof twenty birds per treatment.

In ovo inoculation had no impact on hatch rate, since 97.5% ofuninoculated eggs and 98.8% of inoculated eggs hatched. Both groups weresimilar in weight at hatch and gained weight at a similar ratethroughout the study. The two routes of administration of C. jejuni tobroiler chicks resulted in equivalent colonization both in terms of thenumber of birds colonized and the number of C. jejuni per gram of cecalcontents. No signs of clinical significance were observed in any of thebirds. These data indicate that in ovo inoculation of broiler chickswith C. jejuni is as safe as per os inoculation.

The geometric mean antibody titer in response to in ovo inoculation wasat least as great as, if not greater than, the titer resulting from peros inoculation at day of hatch. Therefore, not only can wild type C.jejuni UA535 be safely administered in ovo to broiler chicks, butadministration by this route results in robust colonization and immuneresponse.

EXAMPLE 2

To demonstrate that colonization after in ovo inoculation with C. jejuniis not unique to strain UA535, colonization of birds by another humanclinical isolate, C. jejuni 81-176, was evaluated. Groups of broilereggs were injected on Day 18 of embryo incubation with 1.8×10⁵ or1.4×10⁷ CFU of C. jejuni 81-176. A third group of eggs remaineduninoculated. At hatch, a group of chicks from the uninoculated eggswere retained as uninoculated control birds, and a second group ofchicks from these eggs were given 8.3×10⁶ CFU of C. jejuni 81-176orally.

The data set obtained demonstrated that in ovo administration of C.jejuni strain 81-176 is at least as effective at colonizing broilerchicks as oral administration on the day of hatch.

EXAMPLE 3

On day 18 of embryo incubation, a group of broiler chicken eggs wereinoculated in ovo with 7×10⁷ CFU of C. jejuni CsrA. This strain has amutation in CsrA, a protein involved in global regulation of carbonstorage. A second group of eggs were retained as uninoculated controls.At hatch, a group of chicks from the uninoculated eggs were given 3×10⁸CFU of C. jejuni CsrA orally. Another group of chicks hatched fromuninoculated eggs were retained as controls.

The results indicated that the mutant C. jejuni strain colonized all ofthe birds when administered in ovo, yet colonized only 3 out of 6 birdswhen given orally. Also, the mutant was able to fully colonize birds andinduce an antibody response when administered in ovo, but appeared to beless effective at colonizing birds when given by the oral route on theday of hatch.

EXAMPLE 4

On day 18 of embryo incubation, groups of broiler chicken eggs wereinoculated in ovo with 9×10⁶ CFU of C. jejuni HspR, 8×10⁷ CFU of C.jejuni HtrA, or 6×10⁷ CFU of C. jejuni Dps. The HspR strain has amutation in a heat shock protein; the HtrA strain has a mutation in aserine protease gene; and the Dps strain is mutated in a gene involvedin iron acquisition. Another group of eggs was retained as uninoculatedcontrols. At hatch, groups of chicks from the uninoculated eggs weregiven 4×10⁶ CFU of C. jejuni HspR, 1×10⁸ CFU of C. jejuni HtrA, or 1×10⁸CFU of C. jejuni Dps orally. Another group of chicks hatched fromuninoculated eggs was retained as controls.

The HspR mutant was undetectable in samples from birds inoculated peros, but half the birds given this strain in ovo were colonized with C.jejuni. The results indicate that this mutant was able to colonizebroiler chicks after in ovo administration, but not after oraladministration on the day of hatch. The other mutants readily colonizedbirds and induced an immunological response regardless of route ofadministration.

EXAMPLE 5

On day 18 of embryo incubation, groups of broiler chicken eggs wereinoculated in ovo with 7.3×10⁷ CFU of C. jejuni flbA, 1.1×10⁸ CFU of C.jejuni Pnp, or 1.2×10⁸ CFU of C. jejuni SurE. The FlbA strain is mutatedin a gene encoding for a flagellar structural protein; the Pnp strain ismutated in a gene encoding for a nucleotidyltransferase; and the SurEstrain is mutated in a gene encoding a phosphatase. Another group ofeggs was retained as uninoculated controls. At hatch, groups of chicksfrom the uninoculated eggs were given 9.3×10⁷ CFU of C. jejuni flbA,1.6×10⁸ CFU of C. jejuni Pnp, or 8×10⁷ CFU of C. jejuni SurE orally.Another group of chicks hatched from uninoculated eggs was retained ascontrols.

The FlbA mutant demonstrated a transient colonization when given by thein ovo route, and this mutant appeared unable to colonize by the oralroute. The Pnp mutant colonized birds effectively regardless of route ofadministration. The SurE mutant colonized birds effectively whenadministered by the in ovo route, but was unable to colonize birds whenadministered orally.

EXAMPLE 6

On Day 18 of incubation, a group of broiler chicken eggs were injectedin ovo with 2×10⁶ cells of wild-type C. jejuni strain UA535. A secondgroup of eggs were maintained as uninoculated controls. At hatch, agroup of birds from uninoculated eggs were inoculated per os with 2×10⁶cells of C. jejuni UA535. The remaining birds that hatched fromuninoculated eggs were kept as controls. At 27 days post-hatch, half ofthe birds in each group were placed on medicated water containing 100mg/L kanamycin to eliminate the immunizing C. jejuni strain. Theseinfected/cleared birds would therefore be considered “immunized”.

At 34 days post-hatch, selected groups of birds were exposed to birdspreviously infected with C. jejuni CjM20, a strain genetically-modifiedto be resistant to kanamycin. This method allowed the spread of CjM20from the infected birds to the “immunized” birds. By diagnostic means,it was possible to distinguish the strain CjM20 from the immunizingstrain UA535 in the samples collected.

Non-immunized, non-medicated birds challenged with CjM20 were fullycolonized by 41 days of age. Colonization was delayed somewhat bykanamycin treatment, with about half the birds colonized by Day 41. Allof the non-immunized, medicated, challenged birds were fully colonizedby 49 days of age. Both groups of immunized birds had reduced numbers ofC. jejuni compared with their non-immunized counterparts. All recoveredbacteria appeared resistant to kanamycin, indicating that the immunizingstrain was eliminated by kanamycin treatment. Immunized, non-challengedbirds remained free of C. jejuni throughout the study. Thus,immunization of broiler chicks against colonization with C. jejuni wasat least as effective by the in ovo route as by the oral route.

1. A method of inducing a protective immune response in a bird againstCampylobacter jejuni, comprising administering, in ovo, during the finalquarter of incubation, an immunizing effective amount of live cells ofCampylobacter jejuni, wherein said live cells are free of neutralizingantibodies or neutralizing antibody fragments.
 2. The method of claim 1,wherein said bird is a domesticated bird.
 3. The method of claim 2,wherein said domesticated bird is selected from the group consisting ofa chicken, a turkey, and a duck.
 4. The method of claim 1, wherein thelive cells are wild type or have been modified genetically.
 5. Themethod of claim 4, wherein a heterologous polynucleotide sequence hasbeen introduced into the live cells of Campylobacter jejuni.
 6. Themethod of claim 5, wherein said heterologous polynucleotide sequenceencodes a protein essential in colonization of a domesticated bird byCampylobacter jejuni.
 7. The method of claim 5, wherein saidheterologous polynucleotide sequence encodes an antigen from a virus,bacteria, or parasite that causes disease in a domesticated bird.
 8. Themethod of claim 5, wherein said heterologous polynucleotide sequenceencodes an antigen from an organism that causes food-borne illness inhumans.
 9. The method of claim 5, wherein said heterologouspolynucleotide sequence encodes a protein that enhances the growth orfeed efficiency of a domesticated bird.
 10. The method of claim 5,wherein said heterologous polynucleotide sequence encodes a protein thatstimulates the bird's immune system.
 11. The method of claim 1, furthercomprising administering a veterinary-acceptable carrier.
 12. The methodof claim 11, wherein said veterinary-acceptable carrier is combined withthe live cells of Campylobacter jejuni prior to in ovo administration.13. The method of claim 11, wherein said veterinary-acceptable carrieris administered to the bird in feed or water, or by aerosol spray, atany time after hatching.
 14. The method of claim 12, wherein saidveterinary-acceptable carrier is an adjuvant.
 15. The method of claim14, wherein said adjuvant has an immune-stimulating activity.
 16. Themethod of claim 1, wherein live cells of Campylobacter jejuni arecombined with at least one other immunogen selected from a viral, abacterial or a protozoan immunogen.
 17. The method of claim 13, whereinsaid veterinary-acceptable carrier is an adjuvant.
 18. The method ofclaim 17, wherein said adjuvant has an immune-stimulating activity. 19.A method of inducing a protective immune response in a bird againstCampylobacter jejuni, comprising administering, in ovo, during the finalquarter of incubation, an immunizing effective amount of live cells ofCampylobacter jejuni, wherein said live cells are free of neutralizingantibodies or neutralizing antibody fragments; and wherein said bird isthen harvested for human food consumption.